G-quadruplexes in helminth parasites.

Parasitic helminths infecting humans are highly prevalent infecting ∼2 billion people worldwide, causing inflammatory responses, malnutrition and anemia that are the primary cause of morbidity. In addition, helminth infections of cattle have a significant economic impact on livestock production, milk yield and fertility. The etiological agents of helminth infections are mainly Nematodes (roundworms) and Platyhelminths (flatworms). G-quadruplexes (G4) are unusual nucleic acid structures formed by G-rich sequences that can be recognized by specific G4 ligands. Here we used the G4Hunter Web Tool to identify and compare potential G4 sequences (PQS) in the nuclear and mitochondrial genomes of various helminths to identify G4 ligand targets. PQS are nonrandomly distributed in these genomes and often located in the proximity of genes. Unexpectedly, a Nematode, Ascaris lumbricoides, was found to be highly enriched in stable PQS. This species can tolerate high-stability G4 structures, which are not counter selected at all, in stark contrast to most other species. We experimentally confirmed G4 formation for sequences found in four different parasitic helminths. Small molecules able to selectively recognize G4 were found to bind to Schistosoma mansoni G4 motifs. Two of these ligands demonstrated potent activity both against larval and adult stages of this parasite.

Letter to the Editor: Significant mutation enrichment in inverted repeat sites of new SARS-CoV-2 strains.

In a recently published paper, we have found that SARS-CoV-2 hot-spot mutations are significantly associated with inverted repeat loci and CG dinucleotides. However, fast-spreading strains with new mutations (so-called mink farm mutations, England mutations and Japan mutations) have been recently described. We used the new datasets to check the positioning of mutation sites in genomes of the new SARS-CoV-2 strains. Using an open-access Palindrome analyzer tool, we found mutations in these new strains to be significantly enriched in inverted repeat loci.

SARS-CoV-2 hot-spot mutations are significantly enriched within inverted repeats and CpG island loci.

SARS-CoV-2 is an intensively investigated virus from the order Nidovirales (Coronaviridae family) that causes COVID-19 disease in humans. Through enormous scientific effort, thousands of viral strains have been sequenced to date, thereby creating a strong background for deep bioinformatics studies of the SARS-CoV-2 genome. In this study, we inspected high-frequency mutations of SARS-CoV-2 and carried out systematic analyses of their overlay with inverted repeat (IR) loci and CpG islands. The main conclusion of our study is that SARS-CoV-2 hot-spot mutations are significantly enriched within both IRs and CpG island loci. This points to their role in genomic instability and may predict further mutational drive of the SARS-CoV-2 genome. Moreover, CpG islands are strongly enriched upstream from viral ORFs and thus could play important roles in transcription and the viral life cycle. We hypothesize that hypermethylation of these loci will decrease the transcription of viral ORFs and could therefore limit the progression of the disease.

Electrochemistry of Cobalta Bis(dicarbollide) Ions Substituted at Carbon Atoms with Hydrophilic Alkylhydroxy and Carboxy Groups.

In this study we explore the effect on the electrochemical signals in aqueous buffers of the presence of hydrophilic alkylhydroxy and carboxy groups on the carbon atoms of cobalta bis(dicarbollide) ions. The oxygen-containing exo-skeletal substituents of cobalta bis(dicarbollide) ions belong to the perspective building blocks that are considered for bioconjugation. Carbon substitution provides wider versatility and applicability in terms of the flexibility of possible chemical pathways. However, until recently, the electrochemistry of compounds substituted only on boron atoms could be studied, due to the unavailability of carbon-substituted congeners. In the present study, electrochemistry in aqueous phosphate buffers is considered along with the dependence of electrochemical response on pH and concentration. The compounds used show electrochemical signals around -1.3 and +1.1 V of similar or slightly higher intensities than in the parent cobalta bis(dicarbollide) ion. The signals at positive electrochemical potential correspond to irreversible oxidation of the boron cage (the C2B9 building block) and at negative potential correspond to the reversible redox process of (CoIII/CoII) at the central atom. Although the first signal is typically sharp and its potential can be altered by a number of substituents, the second signal is complex and is composed of three overlapping peaks. This signal shows sigmoidal character at higher concentrations and may be used as a diagnostic tool for aggregation in solution. Surprisingly enough, the observed effects of the site of substitution (boron or carbon) and between individual groups on the electrochemical response were insignificant. Therefore, the substitutions would preserve promising properties of the parent cage for redox labelling, but would not allow for the further tuning of signal position in the electrochemical window.

G-quadruplexes in H1N1 influenza genomes.

BACKGROUND: Influenza viruses are dangerous pathogens. Seventy-Seven genomes of recently emerged genotype 4 reassortant Eurasian avian-like H1N1 virus (G4-EA-H1N1) are currently available. We investigated the presence and variation of potential G-quadruplex forming sequences (PQS), which can serve as targets for antiviral treatment. RESULTS: PQS were identified in all 77 genomes. The total number of PQS in G4-EA-H1N1 genomes was 571. Interestingly, the number of PQS per genome in individual close relative viruses varied from 4 to 12. PQS were not randomly distributed in the 8 segments of the G4-EA-H1N1 genome, the highest frequency of PQS being found in the NP segment (1.39 per 1000 nt), which is considered a potential target for antiviral therapy. In contrast, no PQS was found in the NS segment. Analyses of variability pointed the importance of some PQS; even if genome variation of influenza virus is extreme, the PQS with the highest G4Hunter score is the most conserved in all tested genomes. G-quadruplex formation in vitro was experimentally confirmed using spectroscopic methods. CONCLUSIONS: The results presented here hint several G-quadruplex-forming sequences in G4-EA-H1N1 genomes, that could provide good therapeutic targets.

Carborane- or Metallacarborane-Linked Nucleotides for Redox Labeling. Orthogonal Multipotential Coding of all Four DNA Bases for Electrochemical Analysis and Sequencing.

We report a series of 2'-deoxyribonucleoside triphosphates bearing dicarba-nido-undecaborate ([C2B9H11]1-), [3,3'-iron-bis(1,2-dicarbollide)]- (FESAN, [Fe(C2B9H11)2]2-) or [3,3'-cobalt-bis(1,2-dicarbollide)]- (COSAN, [Co(C2B9H11)2]2-) groups prepared either through the Sonogashira cross-coupling or the CuAAC click reaction. The modified dNXTPs were substrates for KOD XL DNA polymerase in enzymatic synthesis of modified DNA through primer extension (PEX). The nido-carborane- and FESAN-modified nucleotides gave analytically useful oxidation signals in square-wave voltammetry and were used for redox labeling of DNA. The redox-modified DNA probes were prepared by PEX using tailed primers and were hybridized to electrode (gold or glassy carbon) containing capture oligonucleotides. The combination of nido-carborane- and FESAN-linked nucleotides with 7-ferrocenylethynyl-7-deaza-dATP and 7-deaza-dGTP allowed polymerase synthesis of DNA fully modified at all four nucleobases, and each of the redox labels gave four differentiable and ratiometric signals in voltammetry. Thus, the combination of these four redox labels constitutes the first fully orthogonal redox coding of all four canonical nucleobases, which can be used for determination of nucleobase composition of short DNA stretches in one simple PEX experiment with electrochemical readout.

Structures and stability of simple DNA repeats from bacteria.

DNA is a fundamentally important molecule for all cellular organisms due to its biological role as the store of hereditary, genetic information. On the one hand, genomic DNA is very stable, both in chemical and biological contexts, and this assists its genetic functions. On the other hand, it is also a dynamic molecule, and constant changes in its structure and sequence drive many biological processes, including adaptation and evolution of organisms. DNA genomes contain significant amounts of repetitive sequences, which have divergent functions in the complex processes that involve DNA, including replication, recombination, repair, and transcription. Through their involvement in these processes, repetitive DNA sequences influence the genetic instability and evolution of DNA molecules and they are located non-randomly in all genomes. Mechanisms that influence such genetic instability have been studied in many organisms, including within human genomes where they are linked to various human diseases. Here, we review our understanding of short, simple DNA repeats across a diverse range of bacteria, comparing the prevalence of repetitive DNA sequences in different genomes. We describe the range of DNA structures that have been observed in such repeats, focusing on their propensity to form local, non-B-DNA structures. Finally, we discuss the biological significance of such unusual DNA structures and relate this to studies where the impacts of DNA metabolism on genetic stability are linked to human diseases. Overall, we show that simple DNA repeats in bacteria serve as excellent and tractable experimental models for biochemical studies of their cellular functions and influences.

Global analysis of inverted repeat sequences in human gene promoters reveals their non-random distribution and association with specific biological pathways.

Inverted repeats (IR) play important roles in specific DNA-dependent processes in simple prokaryotes to complex eukaryotes. They are recognized by a variety of proteins including restriction enzymes, helicases and transcription factors. We evaluate the presence and localization of IRs in all validated human promoter sequences within 1000 bp upstream and downstream of the transcription start site (TSS). The occurrence of 7 bp and longer IRs is located non-randomly in promoter regions, with enrichment within 200 bp upstream of the TSS. The highest frequency of IRs is just before TSS for repeats of 8 bp or longer. A comparison of promoters divided according to the occurrence of five individual promoter motifs shows unique location patterns of IRs. Principal component analyses and hierarchical clustering of IRs abundance demonstrated that they are depleted and/or not enriched in the promoters of stably expressed genes, but show significant enrichments for specific dynamically regulated biological pathways.

Tracing dsDNA Virus-Host Coevolution through Correlation of Their G-Quadruplex-Forming Sequences.

The importance of gene expression regulation in viruses based upon G-quadruplex may point to its potential utilization in therapeutic targeting. Here, we present analyses as to the occurrence of putative G-quadruplex-forming sequences (PQS) in all reference viral dsDNA genomes and evaluate their dependence on PQS occurrence in host organisms using the G4Hunter tool. PQS frequencies differ across host taxa without regard to GC content. The overlay of PQS with annotated regions reveals the localization of PQS in specific regions. While abundance in some, such as repeat regions, is shared by all groups, others are unique. There is abundance within introns of Eukaryota-infecting viruses, but depletion of PQS in introns of bacteria-infecting viruses. We reveal a significant positive correlation between PQS frequencies in dsDNA viruses and corresponding hosts from archaea, bacteria, and eukaryotes. A strong relationship between PQS in a virus and its host indicates their close coevolution and evolutionarily reciprocal mimicking of genome organization.

Vicinal Diol-Tethered Nucleobases as Targets for DNA Redox Labeling with Osmate Complexes.

Six-valent osmium (osmate) complexes with nitrogenous ligands have previously been used for the modification and redox labeling of biomolecules involving vicinal diol moieties (typically, saccharides or RNA). In this work, aliphatic (3,4-dihydroxybutyl and 3,4-dihydroxybut-1-ynyl) or cyclic (6-oxo-6-(cis-3,4-dihydroxypyrrolidin-1-yl)hex-2-yn-1-yl, PDI) vicinal diols are attached to nucleobases to functionalize DNA for subsequent redox labeling with osmium(VI) complexes. The diol-linked 2'-deoxyribonucleoside triphosphates were used for the polymerase synthesis of diol-linked DNA, which, upon treatment with K2 OsO3 and bidentate nitrogen ligands, gave the desired Os-labeled DNA, which were characterized by means of the gel-shift assay and ESI-MS. Through ex situ square-wave voltammetry at a basal plane pyrolytic graphite electrode, the efficiency of modification/labeling of individual diols was evaluated. The results show that the cyclic cis-diol (PDI) was a better target for osmylation than that of the flexible aliphatic ones (alkyl- or alkynyl-linked). The osmate adduct-specific voltammetric signal obtained for OsVI -treated DNA decorated with PDI showed good proportionality to the number of PDI per DNA molecule. The OsVI reagents (unlike OsO4 ) do not attack nucleobases; thus offering specificity of modification on the introduced glycol targets.

G4Hunter web application: a web server for G-quadruplex prediction.

MOTIVATION: Expanding research highlights the importance of guanine quadruplex structures. Therefore, easy-accessible tools for quadruplex analyses in DNA and RNA molecules are important for the scientific community. RESULTS: We developed a web version of the G4Hunter application. This new web-based server is a platform-independent and user-friendly application for quadruplex analyses. It allows retrieval of gene/nucleotide sequence entries from NCBI databases and provides complete characterization of localization and quadruplex propensity of quadruplex-forming sequences. The G4Hunter web application includes an interactive graphical data representation with many useful options including visualization, sorting, data storage and export. AVAILABILITY AND IMPLEMENTATION: G4Hunter web application can be accessed at: http://bioinformatics.ibp.cz. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Tuning of Oxidation Potential of Ferrocene for Ratiometric Redox Labeling and Coding of Nucleotides and DNA.

Three sets of 7-deazaadenine and cytosine nucleosides and nucleoside triphosphates bearing either unsubstituted ferrocene, octamethylferrocene and ferrocenecarboxamide linked through an alkyne tether to position 7 or 5, respectively, were designed and synthesized. The modified dNFcX TPs were good substrates for KOD XL DNA polymerase in primer extension and were used for enzymatic synthesis of redox-labelled DNA probes. Square-wave voltammetry showed that the octamethylferrocene oxidation potential was shifted to lower values, whilst the ferrocenecarboxamide was shifted to higher potentials, as compared to ferrocene. Tailed PEX products containing different ratios of Fc-labelled A (dAFc ) and FcPa-labelled C (dCFcPa ) were synthesized and hybridized with capture oligonucleotides immobilized on gold electrodes to study the electrochemistry of the redox-labelled DNA. Clearly distinguishable, fully orthogonal and ratiometric peaks were observed for the dAFc and dCFcPa bases in DNA, demonstrating their potential for use in redox coding of nucleobases and for the direct electrochemical measurement of the relative ratio of nucleobases in an unknown sequence of DNA.

Electrochemical Reduction and Oxidation of Eight Unnatural 2'-Deoxynucleosides at a Pyrolytic Graphite Electrode.

Recently we showed the reduction and oxidation of six natural 2'-deoxynucleosides in the presence of the ambient oxygen using the very broad potential window of a pyrolytic graphite electrode (PGE). Using the same procedure, 2'-deoxynucleoside analogs (dNs) that are parts of an artificially expanded genetic information system (AEGIS) were analyzed. Seven of the eight tested AEGIS dNs provided specific signals (voltammetric redox peaks). These signals, described here for the first time, will be used in future work to analyze DNA built from expanded genetic alphabets, helping to further develop AEGIS technology and its applications. Comparison of the electrochemical behavior of unnatural dNs with the previously documented behaviors of natural dNs also provides insights into the mechanisms of their respective redox processes.

The Rich World of p53 DNA Binding Targets: The Role of DNA Structure.

The tumor suppressor functions of p53 and its roles in regulating the cell cycle, apoptosis, senescence, and metabolism are accomplished mainly by its interactions with DNA. p53 works as a transcription factor for a significant number of genes. Most p53 target genes contain so-called p53 response elements in their promoters, consisting of 20 bp long canonical consensus sequences. Compared to other transcription factors, which usually bind to one concrete and clearly defined DNA target, the p53 consensus sequence is not strict, but contains two repeats of a 5'RRRCWWGYYY3' sequence; therefore it varies remarkably among target genes. Moreover, p53 binds also to DNA fragments that at least partially and often completely lack this consensus sequence. p53 also binds with high affinity to a variety of non-B DNA structures including Holliday junctions, cruciform structures, quadruplex DNA, triplex DNA, DNA loops, bulged DNA, and hemicatenane DNA. In this review, we summarize information of the interactions of p53 with various DNA targets and discuss the functional consequences of the rich world of p53 DNA binding targets for its complex regulatory functions.

The Influence of Quadruplex Structure in Proximity to P53 Target Sequences on the Transactivation Potential of P53 Alpha Isoforms.

p53 is one of the most studied tumor suppressor proteins that plays an important role in basic biological processes including cell cycle, DNA damage response, apoptosis, and senescence. The human TP53 gene contains alternative promoters that produce N-terminally truncated proteins and can produce several isoforms due to alternative splicing. p53 function is realized by binding to a specific DNA response element (RE), resulting in the transactivation of target genes. Here, we evaluated the influence of quadruplex DNA structure on the transactivation potential of full-length and N-terminal truncated p53α isoforms in a panel of S. cerevisiae luciferase reporter strains. Our results show that a G-quadruplex prone sequence is not sufficient for transcription activation by p53α isoforms, but the presence of this feature in proximity to a p53 RE leads to a significant reduction of transcriptional activity and changes the dynamics between co-expressed p53α isoforms.

p53 Binds Preferentially to Non-B DNA Structures Formed by the Pyrimidine-Rich Strands of GAA·TTC Trinucleotide Repeats Associated with Friedreich's Ataxia.

Expansions of trinucleotide repeats (TNRs) are associated with genetic disorders such as Friedreich's ataxia. The tumor suppressor p53 is a central regulator of cell fate in response to different types of insults. Sequence and structure-selective modes of DNA recognition are among the main attributes of p53 protein. The focus of this work was analysis of the p53 structure-selective recognition of TNRs associated with human neurodegenerative diseases. Here, we studied binding of full length p53 and several deletion variants to TNRs folded into DNA hairpins or loops. We demonstrate that p53 binds to all studied non-B DNA structures, with a preference for non-B DNA structures formed by pyrimidine (Py) rich strands. Using deletion mutants, we determined the C-terminal DNA binding domain of p53 to be crucial for recognition of such non-B DNA structures. We also observed that p53 in vitro prefers binding to the Py-rich strand over the purine (Pu) rich strand in non-B DNA substrates formed by sequence derived from the first intron of the frataxin gene. The binding of p53 to this region was confirmed using chromatin immunoprecipitation in human Friedreich's ataxia fibroblast and adenocarcinoma cells. Altogether these observations provide further evidence that p53 binds to TNRs' non-B DNA structures.

Phenothiazine-linked nucleosides and nucleotides for redox labelling of DNA.

Nucleosides and 2'-deoxyribonucleoside triphosphates (dNTPs) bearing phenothiazine (PT) attached to a nucleobase (cytosine or 7-deazaadenine) either directly or through an acetylene linker were prepared through Suzuki or Sonogashira cross-coupling and triphosphorylation, and were studied as building blocks for polymerase construction of modified DNA. The directly PT-substituted dNTPs were better substrates for polymerases than the alkyne-linked dNTPs but all of them were used in enzymatic synthesis of DNA using primer extension, nicking enzyme amplification, PCR or 3'-tail labelling by terminal deoxynucleotidyl transferase. The phenothiazine served as an oxidizable redox label (giving two analytically useful signals of oxidation on electrode) for nucleosides and DNA and was also used in orthogonal combination with previously developed benzofurazane or nitrophenyl labels for redox coding of DNA bases. Therefore, the title PT-linked dNTPs are useful additions to the portfolio of nucleotides for enzymatic synthesis of redox-labelled DNA for electrochemical analysis.

Chloroacetamide-Linked Nucleotides and DNA for Cross-Linking with Peptides and Proteins.

Nucleotides, 2'-deoxyribonucleoside triphosphates (dNTPs), and DNA probes bearing reactive chloroacetamido group linked to nucleobase (cytosine or 7-deazadaenine) through a propargyl tether were prepared and tested in cross-linking with cysteine- or histidine-containing peptides and proteins. The chloroacetamide-modifed dNTPs proved to be good substrates for DNA polymerases in the enzymatic synthesis of modified DNA probes. Modified nucleotides and DNA reacted efficiently with cysteine and cysteine-containing peptides, whereas the reaction with histidine was sluggish and low yielding. The modified DNA efficiently cross-linked with p53 protein through alkylation of cysteine and showed potential for cross-linking with histidine (in C277H mutant of p53).

Azidopropylvinylsulfonamide as a New Bifunctional Click Reagent for Bioorthogonal Conjugations: Application for DNA-Protein Cross-Linking.

N-(3-Azidopropyl)vinylsulfonamide was developed as a new bifunctional bioconjugation reagent suitable for the cross-linking of biomolecules through copper(I)-catalyzed azide-alkyne cycloaddition and thiol Michael addition reactions under biorthogonal conditions. The reagent is easily clicked to an acetylene-containing DNA or protein and then reacts with cysteine-containing peptides or proteins to form covalent cross-links. Several examples of bioconjugations of ethynyl- or octadiynyl-modified DNA with peptides, p53 protein, or alkyne-modified human carbonic anhydrase with peptides are given.

G-quadruplex-based structural transitions in 15-mer DNA oligonucleotides varying in lengths of internal oligo(dG) stretches detected by voltammetric techniques.

Electrochemical methods, particularly when applied in connection with mercury-containing electrodes, are excellent tools for studying nucleic acids structure and monitoring structural transitions. We studied the effect of the length of the central (dG) n stretch (varying from 0 to 15 guanine residues) in 15-mer oligodeoxynucleotides (ODN, G0 to G15) on their electrochemical and interfacial behavior at mercury and carbon electrodes. The intensity of guanine oxidation signal at the carbon electrode (peak G(ox)) was observed to increase continuously with number of guanines between 0 and 15, with only a slight positive shift for ODNs with seven or more guanines in the central segment. Very different effects were observed when the peak G(HMDE) was measured at the mercury electrode. Intensity of the latter signal increased with number of guanines up to G5, and decreased sharply with further elongation of the (dG) n stretch. CD spectroscopy and electrophoresis experiments revealed formation of parallel intermolecular quadruplex structures for ODNs containing five or more G residues. Further measurements made by cyclic and alternating-current voltammetry revealed a strong influence of the ODN structure on their behavior at electrically charged surfaces.